The present invention relates to a novel solution for preserving and maintaining organs and portions thereof, in particular heart and myocardial tissue and lung and lung tissue.
Preservation of the viability of donor organs continues to be an important goal in transplantation. Typically the organ to be transplanted must be stored and shipped to the prospective recipient. The ability to prolong the cellular viability of the organ during storage and transportation is very important to the success of the transplant operation. Preservative solutions play an important role in the longevity of the organ. Prior known solutions for organ preservation include those described by Berdyaev et al., U.S. Pat. No. 5,432,053; Belzer et al., U.S. Pat. Nos. 4,798,824, 4,879,283; and 4,873,230; Taylor, U.S. Pat. No. 5,405,742; Dohi et al., U.S. Pat. No. 5,565,317; Stern et al., U.S. Pat. No. 5,370,989 and 5,552,267.
Currently there is no consensus among practitioners regarding an optimal solution for heart preservation. Solutions include those classified as intracellular ([Na++] less than 70 mEq/L) or extracellular ([Na++]xe2x89xa770 mEq/L). A recent survey showed that there were at least 167 organ preservation solutions available for heart transplantation, and that there was significant variation in solution usage observed among major U.S. regions of transplantation activity. (Demmy et al., Organ preservation solutions in heart transplantationxe2x80x94patterns of usage and related survival. Transplantation 63(2): 262-269 (1997)). Presently known solutions for cardiac preservation include those described by Oz et al., Novel Preservation Solution Permits 24-Hour Preservation in Rat and Baboon Cardiac Transplant Models, Circulation 88(2)L291-297 (1993) at Columbia University in New York, and Belzer and Southard in Transplantation 45:673-676 (1988), at the University of Wisconsin. Other solutions for heart preservation and cardioplegia include those disclosed in U.S. Pat. No. 5,407,793 by Del Nido at al. (Univ. Of Pittsburgh); and U.S. Pat. No. 4,938,961 by Collins et al.).
The development of myocardial preservation solutions typically requires the use of whole organ models to assess the performance of such solutions. These methods are animal and labor intensive. In addition such methods rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual solution components difficult. Another problem inherent in the whole organ test model is that individual responses can not be removed as a variable when organs must be harvested from many different donors to be tested.
The use of tissue slices have advantages over both in vivo whole organ models and in vitro cellular models to study organ function. Advantages over whole organ models include a reduction in the number of animals used, a decrease in experimental variation, more rapid production of experimental results and elimination of humoral and neuronal systemic influences. (Fisher et al., Cryobiology 33:163 (1996)). Preservation and homogenization of tissue in a measured, standardized fashion is much simpler using slices rather than whole organs, facilitating the measurement of quantifiable biochemical endpoints. As a result, comparative experiments that typically would take several weeks using whole organs can be done in two days using the slice model.
Compared to cell culture or cell suspension models, tissue slices maintain the multicellular composition of intact tissue, preserving the intercellular connections used in maintaining contact inhibition, signal transmission and hormonal and ion transport (Fisher et al., Cryobiology 33:163 (1996)). These intercellular connections are typically lost with the protease digestion necessary to isolate single cell types used with in vitro models (Fisher et al., supra). In addition, with optimization of the slice thickness, efficient gas and nutrient exchange can be maintained with diffusion into the tissue of entering nutrients and oxygen and egress of cellular byproducts. (Fisher et al., supra). As a result of these physiologic advantages and the efficiencies of the tissue slice model, individual solution components can be readily studied for their contribution to one or more measures of myocardial preservation.
The use of organ slices for in vitro toxicological and metabolic studies of potential drugs and environmental contaminants has been described for various organs including the liver. (Fisher et al., Cryobiology 28: 131-142 (1991); Fisher et al., Cryobiology 30:250-261 (1993); Fisher et al., U.S. Pat. No. 5,328,821 and Gandolfi et al., Toxicology 105:2-3, 2850290 (1995). Heart tissue slices have been used in pharmacology and toxicology tests to evaluate cold and cryopreservation solutions (Parrish et al., Life Sci. 57:21 (1887-901 (1995)).
Assessment of cellular viability is a requirement for determining effectiveness of tissue preservation with cold storage. Preservation of myocardial cellular viability can be assessed by measurement of ATP levels and capacity for protein synthesis. ATP levels are critical in the stored heart for energy production during and following reperfiision. Capacity for protein synthesis is a general indicator of cellular viability because it requires the integration of several complex biochemical pathways.
There remains a need for improved solutions to preserve viability and maintain function of donor organs for transplantation and research.
Accordingly, the invention comprises novel solutions for preserving and maintaining the viability of solid tissue such as organs and portions thereof, particularly heart and lung tissue. The solutions contain a sufficient amount of a cryopreservative agent, anti-oxidant and an energy source to support intracellular function and maintain cellular viability. The cryopreservative agent can be a chain of simple sugars including disaccharides, trisaccharides, or a chain of four or more saccharides; one example is trehalose. The anti-oxidant can be glutathione and/or allopurinol, and the energy source is cyclic AMP, cyclic GMP or adenosine. The solution can further contain cations such as calcium, potassium and magnesium, an anticoagulant such as heparin, a polysaccharide such as dextran, nitroglycerin and at least one amino acid, for example, L-arginine and is at a pH of from 7.0 to 8.0.
An embodiment of the solution of the invention for preservation and maintenance of the viability of heart tissue includes the following components:
from 0.01 g/L to 10 g/L of cations;
from 3 g/L to 100 g/L of a polysaccharide cryopreservative agent;
from 100 to 30,000 units/L of an anticoagulant;
from 25 g/L to 40 g/L of a polysaccharide;
from 10 mg/L to 1000 mg/L of nitroglycerin;
from 0.10 g/L to 10 g/L of an amino acid;
from 0.01 g/L to 10 g/L of an anti-oxidant; and
from 0.01 g/L to 10 g/L of an energy source, at a pH of approximately 7.4.
A preferred embodiment of the solution of the invention for preservation and maintenance of the viability of heart tissue includes the following components:
2.72 g/L potassium phosphate (20 mmol/L)
1.93 g/L magnesium sulfate (15 mmol/L)
0.11 g/L calcium chloride (1 mmol/L)
30.0 g/L trehalose
10,000 units/L heparin
30.0 g/L dextran
100 mg/L nitroglycerin
1.34 g/L adenosine (5 mmol/L)
1.74 g/L L-arginine (10 mmol/L)
0.14 g/L allopurinol (1 mmol/L)
0.92 g/L glutathione (reduced, 3 mmol/L) and
0.98 g/L db-cyclic AMP (2 mmol/L) at a pH of 7.4.
The invention includes a method of preserving and/or maintaining an organ or tissue by contacting it with a solution of the invention, and a method of transplantation by grafting an organ or tissue and perfusing it with the solution of the invention.
Another embodiment of the solution of the invention for preservation and protection of lung tissue includes the following components:
from 0.01 g/L to 10 g/L cations;
from 3 g/L to 100 g/L of a polysaccharide cryopreservative agent;
from 100 units/L to 30,000 units/L of an anticoagulant;
from 25 g/L to 40 g/L of polysaccharide;
from 10 mg/L to 1000 mg/L of nitroglycerin;
from 0.01 g/L to 10 g/L of an antioxidant; and
from 0.01 g/L to 10 g/L of an energy source, at a pH of from 7 to 8.
In a preferred embodiment, the solution of the invention for lung tissue contains magnesium, calcium and potassium ions, the polysaccharide cryopreservative trehalose, the anticoagulant heparin, the polysaccharide dextran, the amino acid arginine, the antioxidant glutathione and/or allopurinol, and cyclic GMP, cyclic AMP or adenosine as the energy source.
In a particularly preferred embodiment of the solution for lung, the solution contains the following components:
2.72 g/L potassium phosphate (20 mmol/L)
1.93 g/L magnesium sulfate (15 mmol/L)
0.11 g/L calcium chloride (1 mmol/L)
30.0 g/L trehalose
10,000 units/L heparin
30.0 g/L dextran
100 mg/L nitroglycerin
1.34 g/L adenosine (5 mmol/L)
0.14 g/L allopurinol (1 mmol/L)
0.92 g/L glutathione (reduced, 3 mmol/L)
89.2 mg/L 8-Bromo-c-GMP (200 mmol/L) and
16.0 mg/L Dexamethasone at a pH of 7.4.